Crystalline forms of gsk1278863, preparation method and pharmaceutical use thereof

ABSTRACT

The present disclosure relates to crystalline form CS1 and CS9 of a hypoxia-inducible factor prolyl hydroxylase inhibitor compound (I) GSK1278863, processes for preparation, and uses for preparing drugs treating and/or preventing anemia thereof.

TECHNICAL FIELD

The present disclosure relates to the field of pharmaceutical chemistry,particularly relates to novel crystalline forms of a hypoxia-induciblefactor prolyl hydroxylase inhibitor, processes for preparation and usethereof.

BACKGROUND

GSK1278863 (Daprodustat) is an oral hypoxia-inducible factor prolylhydroxylase inhibitor developed by GlaxoSmithKline for the treatment ofanemias, such as chronic kidney disease related anemia.

Currently, the standard treatment for chronic kidney disease-relatedanemia is injection of recombinant human erythropoietin. However,injecting macromolecular recombinant human erythropoietin is oftenassociated with cardiovascular safety issues.

GSK1278863 is a small molecular oral hypoxia-inducible factor prolylhydroxylase inhibitor. Inhibition of prolyl hydroxylase enzymes promotesthe production of red blood cells. The red blood cells can carry oxygento where the body needs, so as to achieve the purpose of relievinganemia. The mechanism of GSK1278863 is similar to the physiologicaleffects that occur in the body at high altitudes. In clinical,GSK1278863 shows good efficacy and safety in relieving anemia. Comparedwith recombinant human erythropoietin GSK1278863 can achieve the sameefficacy and higher safety. In addition, the oral dosage form ofGSK1278863 makes it more convenient for patients to take compared withthe injection of macromolecular recombinant human erythropoietin.

The chemical name of GSK1278863 isN-[(1,3-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine(hereinafter referred to as “Compound (I)”), and the structure is shownas follows:

In the field of pharmaceutical research, different crystalline forms ofdrug substances have different colors, melting points, solubility,dissolution profiles, chemical stabilities, mechanical stabilities, etc.These properties can affect the quality, safety and efficacy of drugproducts, which leads to differences in clinical efficacy. Therefore,the research and control of crystalline forms of drug substances havebecome an important part in drug development.

So far, no crystal form of GSK1278863 has been disclosed. CN101505752Bdisclosed the chemical structure and preparation method of GSK1278863.The inventors of the present disclosure repeated the method and obtaineda solid of GSK1278863. The obtained solid has low purity, extremely highimpurity content and high hygroscopicity, which is not suitable formedicine. The inventors of the present disclosure discovered thecrystalline form CS1 and form CS9 of GSK1278863 which show excellentperformances. Compared to the prior art solid, the crystalline form CS1and form CS9 of GSK1278863 of the present disclosure have higher purityand lower hygroscopicity. In addition, the crystalline form CS1 and formCS9 provided by the present disclosure have advantages in stability,degradability, solubility, flowability, and in vitro dissolution informulations, which provides a better choice for the preparation of drugproducts containing GSK1278863 and is of great significance for drugdevelopment.

SUMMARY

The main objective of the present disclosure is to provide novelcrystalline forms of GSK1278863, processes for preparation and usethereof.

According to the objective of the present disclosure, crystalline formCS1 of Compound (I) is provided (hereinafter referred to as Form CS1).

The X-ray powder diffraction pattern of Form CS1 shows characteristicpeaks at 2theta values of 6.4°±0.2°, 7.5°±0.2° and 7.9°±0.2° using CuKαradiation.

Furthermore, the X-ray powder diffraction pattern of Form CS1 shows oneor more characteristic peaks at 2theta values of 17.2°±0.2°, 21.0°±0.2°,24.0°±0.2° and 19.3°±0.2°. Preferably, the X-ray powder diffractionpattern of Form CS1 shows characteristic peaks at 2theta values of17.2°±0.2°, 21.0°±0.2°, 24.0°±0.2° and 19.3°±00.2°.

In a preferred embodiment, the X-ray powder diffraction pattern of FormCS1 shows characteristic peaks at 2theta values of 6.4°±00.2°,7.5°±0.2°, 7.9°±0.2°, 17.2°±0.2°, 21.0°±0.2°, 24.0°±0.2°, and 19.3°±0.2°using CuKα radiation.

Without any limitation being implied, in a specific example of thepresent disclosure, the X-ray powder diffraction pattern of Form CS1 issubstantially as depicted in FIG. 1 .

According to the objective of the present disclosure, a process forpreparing Form CS1 is also provided. The process comprises:

-   -   (1) Dissolving GSK1278863 into a solvent selected from the group        consisting of cyclic ethers and ketones, evaporating the        obtained solution at 10-50° C. for crystallization; or    -   (2) Dissolving GSK1278863 into a solvent of cyclic ethers,        adding an anti-solvent for crystallization, separating and        drying to obtain Form CS1.

Furthermore, in method (1), said cyclic ether is preferablytetrahydrofuran; said ketone is preferably acetone, methyl isobutylketone or mixtures thereof; said evaporation temperature is preferablyroom temperature or 50° C.

Furthermore, in method (1), said ketone is more preferably acetone ormethyl isobutyl ketone.

Furthermore, in method (2), said cyclic ether is preferably 1,4-dioxane;said anti-solvent is preferably water; said crystallization time is0.5-24 h.

Furthermore, in method (2), said crystallization time is preferably 2 h.

According to the objective of the present disclosure, crystalline formCS9 of Compound (I) is provided (hereinafter referred to as Form CS9).

The X-ray powder diffraction pattern of Form CS9 shows characteristicpeaks at 2theta values of 4.6°±0.2°, 6.6°±0.2° and 21.1°±0.2° using CuKαradiation.

Furthermore, the X-ray powder diffraction pattern of Form CS9 shows oneor more characteristic peaks at 2theta values of 9.4°±0.2°, 20.2°±0.2°and 24.2°±0.2°.

Preferably, the X-ray powder diffraction pattern of Form CS9 showscharacteristic peaks at 2theta values of 9.4°±0.2°, 20.2°±0.2° and24.2°±0.2°.

In a preferred embodiment, the X-ray powder diffraction pattern of FormCS9 shows characteristic peaks at 2theta values of 4.6°±0.2°, 6.6°±0.2°,21.1°±0.2°, 9.4°±0.2°, 20.2° 00.2° and 24.2° 00.2° using CuKα radiation.

Without any limitation being implied, in a specific example of thepresent disclosure, the X-ray powder diffraction pattern of Form CS9 issubstantially as depicted in FIG. 6 .

According to the objective of the present disclosure, a process forpreparing Form CS9 is also provided. The process comprises:

-   -   (1) Dissolving GSK1278863 into a solvent of ethers and adding        polymer, evaporating the solution at 10-70° C. for        crystallization; or    -   (2) Dissolving GSK1278863 into a solvent mixture of esters and        alcohols, and evaporating the solution at 10-70° C. for        crystallization.

Furthermore, in method (1), said ethers is preferably methyl tert-butylether; said polymer is preferably composed of polycaprolactone,polyoxyethylene, polymethyl methacrylate, hydroxyethyl cellulose, andsodium alginate of equal mass; said evaporation temperature ispreferably 50° C.; Furthermore, in method (2), said ester is preferablyethyl acetate; said alcohol is preferably ethanol; said volume ratio ofester and alcohol is 1:10-10:1; said evaporation temperature ispreferably 50° C.

Furthermore, in method (2), said volume ratio of ester and alcohol ispreferably 1:1.

In the process for preparing Form CS1 and Form CS9 of the presentdisclosure:

-   -   Said “room temperature” is not a specific value, and refers to        10-30° C.

According to the present disclosure, GSK1278863 as a raw material issolid (crystalline and amorphous), semisolid, wax or oil. Preferably,said compound (I) as a raw material is a solid powder.

In the present disclosure, “crystal” or “crystalline form” refers to thesolid being identified by the X-ray diffraction pattern shown herein.Those skilled in the art are able to understand that physicochemicalproperties discussed herein can be characterized. The experimentalerrors depend on the instrument conditions, the sampling processes andthe purity of samples. In particular, those skilled in the art generallyknow that the X-ray diffraction pattern typically varies with theexperimental conditions. It is necessary to point out that, the relativeintensity of the diffraction peaks in the X-ray diffraction pattern mayalso vary with the experimental conditions; therefore, the order of thediffraction peak intensities cannot be regarded as the sole or decisivefactor. In fact, the relative intensity of the diffraction peaks in theX-ray powder diffraction pattern is related to the preferred orientationof the crystals, and the diffraction peak intensities shown herein areillustrative and identical diffraction peak intensities are notrequired. In addition, the experimental error of the diffraction peakposition is usually 5% or less, and the error of these positions shouldalso be taken into account. An error of ±0.2° is usually allowed. Inaddition, due to experimental factors such as sample thickness, theoverall offset of the diffraction peak is caused, and a certain offsetis usually allowed. Thus, it will be understood by those skilled in theart that a crystalline form of the present disclosure is not necessarilyto have the exactly same X-ray diffraction pattern of the example shownherein. As used herein, “the same XRPD pattern” does not mean absolutelythe same, the same peak positions may differ by ±0.2° and the peakintensity allows for some variability. Any crystalline forms whose X-raydiffraction patterns have the same or similar characteristic peaksshould be within the scope of the present disclosure. Those skilled inthe art can compare the patterns shown in the present disclosure withthat of an unknown crystalline form in order to identify whether thesetwo groups of patterns reflect the same or different crystalline forms.

In some embodiments, Form CS1 and Form CS9 of the present disclosure ispure and substantially free of any other crystalline forms. In thepresent disclosure, the term “substantially free” when used to describea novel crystalline form, it means that the content of other crystallineforms in the novel crystalline form is less than 20% (w/w), specificallyless than 10% (w/w), more specifically less than 5% (w/w) and furthermore specifically less than 1% (w/w).

It should be noted that the number and the number range should not beunderstood as the number or number range themselves only. It should beunderstood by those skilled in the art that the specific number can beshifted at specific technical environment without departing from thespirit and principle of the present disclosure.

In the present disclosure, the number of shift ranges expected by one ofskilled in the art is represented by the term “about”.

Crystalline forms of the present disclosure also have the followingadvantages:

-   -   (1) Compared with the prior art, crystalline forms of the        present disclosure have lower hygroscopicity. The weight gain of        the prior art solid at 80% RH is 1.14%. The weight gains of Form        CS1 and Form CS9 at 80% RH are 0.53% and 0.22%, respectively,        which indicates that Form CS1 and Form CS9 can hardly absorb        water and gain weight. Form CS1 and Form CS9 are stable under        high humidity conditions, which is conducive to long-term        storage of drug substances and preparation of drug products;    -   (2) Compared with prior art, crystalline forms of the present        disclosure have higher purity. The purity of the prior art solid        is 81.06%, and the impurity content is high. In a specific        embodiment, the purity of crystalline forms of the present        disclosure is higher than 99%. In another specific embodiment,        the purity of crystalline forms of the present disclosure is        higher than 99.5%;    -   (3) Form CS1 and Form CS9 of the present disclosure have good        stability. The crystalline of Form CS1 and Form CS9 drug        substance doesn't change for at least 3 months when stored under        the conditions of 25° C./60% RH, 40° C./75% RH and 60° C./5% RH.        The chemical purity remains substantially unchanged during        storage. After Form CS1 and Form CS9 are mixed with the        excipients to form drug products, and stored under the condition        of 40° C./75% RH, the crystalline form of Form CS1 and Form CS9        in the drug products doesn't change for at least 1 months.        Crystalline forms of the present disclosure has good stability,        ensuring consistent and controllable quality of the drug        substance and drug products, which is of great significance for        ensuring the efficacy and safety of drugs, avoiding the toxicity        caused by impurities, and preventing the occurrence of adverse        drug reactions;    -   (4) The crystalline forms of the present disclosure have good        solubility in simulated biological media and pure water,        ensuring good dissolution of the crystalline drug substance in        drug products. Higher solubility is beneficial to the effective        absorption of active ingredients in the drug products in the        human body, so as to achieve the ideal drug bioavailability and        efficacy.    -   (5) After being made into tablet formulation, the crystalline        forms of the present invention have good dissolution and        dissolution rate. In pH=6.8 phosphate buffer solution, the        average dissolution at 10 minutes is up to 67.7%, and the        average dissolution at 60 minutes is up to 95.2%. Good in vitro        dissolution is conducive to increasing the degree of drug        absorption and ensure better in vivo exposure, thereby improving        drug's bioavailability and efficacy. High dissolution rate is        beneficial for the drug to achieve peak concentration in plasma        quickly after administration, thus ensuring rapid drug action.

Furthermore, a pharmaceutical composition is provided, saidpharmaceutical composition comprises a therapeutically effective amountof Form CS1 or Form CS9 or combinations thereof and pharmaceuticallyacceptable carriers, diluents or excipients.

Furthermore, Form CS1 or Form CS9 or combinations thereof for the use inpreparing hypoxia-inducible factor prolyl hydroxylase inhibitor drugs.

Furthermore, Form CS1 or Form CS9 or combinations thereof can be usedfor preparing drugs treating and/or preventing anemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an XRPD pattern of Form CS1 according to example 1.

FIG. 2 shows a ¹H NMR spectrum of Form CS1 according to example 1.

FIG. 3 shows a DSC curve of Form CS1 according to example 1.

FIG. 4 shows a TGA curve of Form CS1 according to example 1.

FIG. 5 shows an XRPD pattern of Form CS1 according to example 2.

FIG. 6 shows an XRPD pattern of Form CS9 according to example 5.

FIG. 7 shows a ¹H NMR spectrum of Form CS9 according to example 5.

FIG. 8 shows a DSC curve of Form CS9.

FIG. 9 shows a TGA curve of Form CS9.

FIG. 10 shows a DVS plot of Form CS1.

FIG. 11 shows a DVS plot of Form CS9.

FIG. 12 shows a DVS plot of the solid of the prior art.

FIG. 13 shows an XRPD pattern overlay of Form CS1 of the presentdisclosure before and after being stored under 25° C./60% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 14 shows an XRPD pattern overlay of Form CS1 of the presentdisclosure before and after being stored under 40° C./75% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 15 shows an XRPD pattern overlay of Form CS1 of the presentdisclosure before and after being stored under 60° C./75% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 16 shows an XRPD pattern overlay of Form CS9 of the presentdisclosure before and after being stored under 25° C./60% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 17 shows an XRPD pattern overlay of Form CS9 of the presentdisclosure before and after being stored under 40° C./75% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 18 shows an XRPD pattern overlay of Form CS9 of the presentdisclosure before and after being stored under 60° C./75% RH for 3months (from top to bottom: XRPD pattern before storage, XRPD patternafter storage).

FIG. 19 shows a dissolution profile of a tablet formulation containingForm CS1 of the present disclosure in pH=6.8 phosphate buffer solution.

DETAILED DESCRIPTION

The present disclosure is further illustrated by the following exampleswhich describe the preparation and use of the crystalline forms of thepresent disclosure in detail. It is obvious to those skilled in the artthat many changes in the materials and methods can be accomplishedwithout departing from the scope of the present disclosure.

The abbreviations used in the present disclosure are explained asfollows:

-   -   XRPD: X-ray Powder Diffraction    -   DSC: Differential Scanning Calorimetry    -   TGA: Thermal Gravimetric Analysis    -   ¹H NMR: Proton Nuclear Magnetic Resonance    -   DMSO: Dimethyl sulfoxide

Instruments and Methods Used for Data Collection

X-ray powder diffraction patterns in the present disclosure wereacquired by a Panalytical Empyrean X-ray powder diffractometer. Theparameters of the X-ray powder diffraction method of the presentdisclosure were as follows:

-   -   X-ray Reflection: Cu, Kα    -   Kα1 (Å): 1.540598; Kα2 (Å): 1.544426    -   Kα2/Kα1 intensity ratio: 0.50    -   Voltage: 45 (kV)    -   Current: 40 (mA)    -   Scan range: from 3.0 degree to 40.0 degree

Differential scanning calorimetry (DSC) data in the present disclosurewere acquired by a TA Q2000. The parameters of the DSC method of thepresent disclosure were as follows:

-   -   Heating rate: 10° C./min    -   Purge gas: nitrogen

Thermal gravimetric analysis (TGA) data in the present disclosure wereacquired by a TA Q500. The parameters of the TGA method of the presentdisclosure were as follows:

-   -   Heating rate: 10° C./min    -   Purge gas: nitrogen

Proton nuclear magnetic resonance spectrum data (¹H NMR) were collectedfrom a Bruker Avance II DMX 400M HZ NMR spectrometer. 1-5 mg of samplewas weighed, and dissolved in 0.5 mL of deuterated dimethyl sulfoxide toobtain a solution with a concentration of 2-10 mg/mL.

High Performance Liquid Chromatography (HPLC) data of purity test in thepresent disclosure were collected from an Agilent 1260 with UV VariableWavelength Detector (VWD). The HPLC method parameters for purity test inthe present disclosure are as follows:

-   -   Column: Waters XBridge C8, 150×4.6 mm, 3.5 μm    -   Mobile Phase: A: 0.1% TFA in H₂O        -   B: 0.1% TFA in Acetonitrile    -   Gradient:

Time (min) % B 0.0 20 5.0 50 30.0 80 35.0 80 35.1 20 40.0 20

-   -   Flow rate: 1.0 mL/min    -   Injection Volume: 3μL    -   Column Temperature: 40° C.    -   Diluent: MeOH        -   High Performance Liquid Chromatography (HPLC) data of            solubility test in the present disclosure were collected            from an Agilent 1260 with UV Variable Wavelength Detector            (VWD). The HPLC method parameters are as follows:    -   The HPLC method parameters for solubility test in the present        disclosure are as follows:        -   Column: Waters XBridge C8, 150×4.6 mm, 5 μm        -   Mobile Phase: A: 0.1% TFA in H₂O            -   B: 0.1% TFA in Acetonitrile    -   Gradient:

Time (min) % B 0.0 65 10.0 80 12.0 80 12.1 65 16.0 65

-   -   Flow rate: 1.1 mL/min    -   Injection Volume: 5 μL    -   Column Temperature: 40° C.

Diluent: MeOH

Unless otherwise specified, the following examples were conducted atroom temperature.

Raw materials of GSK1278863 used in the following examples were preparedby known methods in the prior art, for example, the method disclosed inCN101505752B. The prior art solid in the following examples refer to thesolid of GSK1278863 obtained by repeating the preparation methoddisclosed in CN101505752B.

Example 1-4: Preparation of Form CS1 Example 1

6.4 mg of GSK1278863 was weighed and dissolved in 0.5 mL oftetrahydrofuran. The obtained solution was evaporated at roomtemperature for about 2 days to precipitate solid. The obtained solidwas confirmed to be Form CS1. The XRPD pattern is substantially asdepicted in FIG. 1 , and the XRPD data are listed in Table 1.

The ¹H NMR spectrum of Form CS1 is substantially as depicted in FIG. 2 ,and the corresponding data are: ¹HNMR (400 MHz, d₆-DMSO) δ 10.18 (s,1H), 4.62 (s, 2H), 4.11 (d, J=5.8 Hz, 2H), 2.26 (d, J=10.9 Hz, 4H), 1.78(d, J=12.6 Hz, 4H), 1.60 (t, J=11.8 Hz, 6H), 1.27 (d, J=12.9 Hz, 4H),1.13 (s, 2H).

The DSC curve of Form CS1 is substantially as depicted in FIG. 3 . Theendothermic peak at around 242° C. corresponds to the meltingendothermic peak of the Form CS1.

The TGA curve of Form CS1 shows about 0.6% weight loss when heated to150° C., which is substantially as depicted in FIG. 4 .

TABLE 1 Diffraction angle 2θ d spacing Intensity % 3.94 22.44 7.43 5.5315.98 3.05 6.41 13.80 41.18 7.51 11.77 100.00 7.94 11.14 52.84 10.168.71 1.90 12.80 6.91 2.00 13.47 6.57 2.08 15.20 5.83 5.68 15.89 5.582.92 17.15 5.17 15.13 18.52 4.79 3.88 19.25 4.61 13.52 19.92 4.46 7.6020.41 4.35 11.08 20.99 4.23 24.54 22.60 3.93 2.29 24.04 3.70 10.32 26.083.42 2.46 27.19 3.28 2.23 32.89 2.72 0.61

Example 2-3

As shown in Table 2, certain amount of GSK1278863 was weighed anddissolved in corresponding solvents. The obtained solution wasevaporated at 50° C. to obtain solid. The solid obtained in example 2and example 3 was collected and labeled as sample 2 and sample 3. Sample2 and sample 3 were confirmed to be Form CS1.Sample 2 was selected fortests. The XRPD pattern is substantially as depicted in FIG. 5 , and theXRPD data are listed in Table 3.

TABLE 2 Example Amount (mg) Solvent Volume (mL) Label 2 6.7 Methylisobutyl ketone 0.7 2 3 6.6 Acetone 0.7 3

TABLE 3 Diffraction angle 2θ d spacing Intensity % 3.93 22.47 5.30 6.3513.93 37.90 7.53 11.75 100.00 7.92 11.17 41.23 10.12 8.74 1.65 12.806.92 2.37 13.44 6.59 2.83 15.19 5.83 6.22 15.92 5.57 0.81 17.13 5.188.90 18.60 4.77 1.07 19.27 4.61 14.44 19.75 4.50 7.17 20.00 4.44 5.1820.41 4.35 5.27 21.00 4.23 28.87 22.64 3.93 1.35 24.02 3.71 7.91 25.043.56 1.66 26.11 3.41 2.34 27.24 3.27 1.55 28.18 3.17 0.69 28.83 3.100.58 32.76 2.73 0.86 36.60 2.46 0.50

Example 4

10.1 mg of GSK1278863 was weighed and dissolved in 0.5 mL of1,4-dioxane. Then, 2.0 mL of water was added dropwise as ananti-solvent. The obtained solution was stirred at room temperature for2 h, centrifuged, and dried under vacuum to obtain crystalline solid.The obtained solid was confirmed to be Form CS1.

Example 5-6: Preparation of Form CS9 Example 5

4.6 mg of GSK1278863 was weighed and dissolved in 0.7 mL methyltert-butyl ether solvent, followed by adding polymer. The polymer wascomposed of polycaprolactone, polyoxyethylene, polymethyl methacrylate,hydroxyethyl cellulose, and sodium alginate of equal masses. Thesolution was evaporated at 50° C. for about 1 day to precipitate solid.The obtained solid was confirmed to be Form CS9. The XRPD pattern issubstantially as depicted in FIG. 6 , and the XRPD data are listed inTable 4.

The ¹H NMR spectrum of Form CS9 is substantially as depicted in FIG. 7 ,and the corresponding data are: ¹HNMR (400 MHz, d₆-DMSO) δ 10.18 (s,1H), 4.62 (s, 2H), 4.10 (d, J=5.6 Hz, 2H), 2.36-2.17 (m, 4H), 1.78 (d,J=12.4 Hz, 4H), 1.60 (s, 6H), 1.34-1.21 (m, 4H), 1.11 (d, J=13.1 Hz,2H).

TABLE 4 Diffraction angle 2θ d spacing Intensity % 4.58 19.29 19.65 6.5613.47 100.00 9.37 9.44 13.93 10.50 8.43 4.37 13.34 6.64 2.86 15.10 5.871.57 17.13 5.18 4.23 18.51 4.79 1.99 19.54 4.54 4.14 20.18 4.40 17.6521.14 4.20 15.46 24.23 3.67 7.74 30.20 2.96 0.63

Example 6

6.9 mg of GSK1278863 raw materials was weighed and dissolved in 0.7 mLof ethyl acetate: ethanol (V/V, 1:1). The obtained solution wasevaporated at 50° C. for about 4 days to precipitate solid. The obtainedsolid was confirmed to be Form CS9.

The DSC curve of Form CS9 is substantially as depicted in FIG. 8 . Thefirst endothermic peak appears when heated to around 145° C. and thesecond endothermic peak appears when heated to around 237° C.

The TGA curve of Form CS9 shows about 0.2% weight loss when heated to150° C., which is substantially as depicted in FIG. 9 .

Example 7: Hygroscopicity Comparison of Form CS1. Form CS9 and the PriorArt Mini

Dynamic vapor sorption (DVS) was applied to test hygroscopicity of FormCS1, Form CS9 and the prior art solid with about 10 mg of samples under25° C. The results are listed in Table 5. The DVS plots of Form CS1,Form CS9 and the prior art solid are substantially as depicted in FIG.10 , FIG. 11 and FIG. 12 .

TABLE 5 Comparison of hygroscopicity Weight gain under 80% Form RelativeHumidity Form CS1 0.53% Form CS9 0.22% The prior art solid 1.14%

The result indicates that under 25° C./80% RH, Form CS1 and Form CS9 canhardly absorb water and gain weight. Form CS1 and Form CS9 are verystable at high humidity conditions and not hygroscopic. In comparison,the prior art solid shows higher weight gain and higher hygroscopicityunder the same conditions.

Crystalline form with low hygroscopicity doesn't require special dryingconditions during the preparation process, which simplifies thepreparation and post-treatment process of the drug and is easy forindustrial production. Form CS1 and Form CS9 of the present disclosurehave lower hygroscopicity than the prior art solid, which reduces therequirements of the storage environment. For example, it doesn't requirespecial storage humidity, which reduces the cost and is beneficial forlong-term storage of drug substance and drug products.

Example 8: Purity Comparison of Form CS1, Form CS9 and the Prior ArtSolid

HPLC was applied to test the chemical purity of Form CS1, Form CS9 andprior art solid. The results are listed in Table 6.

TABLE 6 Form Form CS1 Form CS9 The prior art solid Purity 99.88% 99.86%81.06% Impurities content  0.12%  0.14% 18.94%

The purity of the drug substance is important for ensuring the efficacyand safety of the drug products and preventing the adverse drugreactions. The impurity content of the prior art solid is extremelyhigh, up to 18.94%, which will lead to significantly lowered activeingredient content or reduced drug activity. High impurity content willalso lead to significantly increased toxicity and side effects of thedrug products. Therefore, the prior art solid cannot be used directly inthe preparation of drug products.

The crystalline forms of the present disclosure have high purity, whichmeets the strict requirements for the purity of the drug substance inthe formulations, and is suitable for the subsequent formulationpreparation and drug production.

Example 9: Stability Assessment of Form CS1

Form CS1 was stored under different conditions of 25° C./60% RH, 40°C./5% RH and 60° C./5% RH. The XRPD pattern overlay before and afterbeing stored is substantially as depicted in FIG. 13 , FIG. 14 and FIG.15 . The results are shown in Table 7.

TABLE 7 Solid form after Initial solid form Condition Time storage FormCS1 25° C./60% RH 3 months Form CS1 (Top of FIG. 13) (Bottom of FIG. 13)Form CS1 40° C./75% RH 3 months Form CS1 (Top of FIG. 14) (Bottom ofFIG. 14) Form CS1 60° C./75% RH 3 months Form CS1 (Top of FIG. 15)(Bottom of FIG. 15)

Furthermore, the inventors also studied the purity change of Form CS1before and after being stored under the conditions of 25° C./60% RH, 40°C./5% RH and 60° C./75% RH for 3 months, and the results are shown inTable 8.

TABLE 8 Purity after Condition Initial purity 3 months Purity change 25°C./60% RH 99.88% 99.78% 0.10% 40° C./75% RH 99.81% 0.07% 60° C./75% RH99.81% 0.07%Form CS1 of the present disclosure doesn't change for at least 3 monthswhen stored under the condition of 25° C./60% RH, 40° C./5% RH and 60°C./5% RH, indicating that Form CS1 has good physical stability. Thechemical purity remains substantially unchanged during storage, whichindicates that Form CS1 does not degrade easily and has good chemicalstability.

As active pharmaceutical ingredient plays an important part in drugproducts, it is vital that the crystalline active pharmaceuticalingredient has good physical and chemical stability. Good physicalstability of Form CS1 avoids crystal transformation during the storageand formulation processes, thereby ensuring consistent and controllablequality of the drug substance and drug products.

During the storage of drug products, the purity decrease will result insignificantly lower drug substance content and reduced drug activity.The decrease in purity also significantly increase the toxicity and sideeffects, affecting the efficacy and safety of drug products. Goodchemical stability of Form CS1 makes the purity keep basically unchangedduring storage, which is of great significance to ensure the efficacyand safety of drugs and prevent the occurrence of adverse drug effects.In addition, stable crystalline form is more controllable in thecrystallization process, and less prone to impurities and mixedcrystals, which is conducive to industrial production.

The stability of drug products containing Form CS1 can be forecastedfrom the stability results of Form CS1 drug substance, providing aguarantee for the preparation of stable drug products.

Example 10: Stability Assessment of Form CS9

Form CS9 was stored under different conditions of 25° C./60% RH, 40°C./5% RH and 60° C./5% RH. The XRPD pattern overlay before and afterbeing stored is substantially as depicted in FIG. 16 , FIG. 17 and FIG.18 . The results are shown in Table 9.

TABLE 9 Solid form after Initial solid form Condition Time storage FormCS9 25° C./60% RH 3 months Form CS9 (Top of FIG. 16) (Bottom of FIG. 16)Form CS9 40° C./75% RH 3 months Form CS9 (Top of FIG. 17) (Bottom ofFIG. 17) Form CS9 60° C./75% RH 3 months Form CS9 (Top of FIG. 18)(Bottom of FIG. 18)

Furthermore, the inventors also studied the purity change of Form CS9before and after being stored under the conditions of 25° C./60% RH, 40°C./5% RH and 60° C./75% RH for 1 month, and the results are shown inTable 10.

TABLE 10 Purity after Condition Initial purity 1 month Purity change 25°C./60% RH 99.88% 99.72% 0.16% 40° C./75% RH 99.78% 0.10% 60° C./75% RH99.76% 0.12%

The crystalline form of Form CS9 of the present disclosure doesn'tchange for at least 3 months and the chemical purity remains for atleast 1 month when stored under the condition of 25° C./60% RH, 40°C./5% RH and 60° C./5% RH, indicating that Form CS9 has good physicalstability. The crystalline of Form CS9 remains substantially unchangedduring storage, which indicates that Form CS9 does not degrade easily,providing a guarantee for the preparation of stable drug products.

Example 11: Kinetic Solubility of Form CS1 and Form CS9

Solubility test method according to the Chinese Pharmacopoeia was usedand different pHs of different organs in human body were considered.According to the above two references, a solvent media set with four pHvalues range from 1.2 to 7.5 were designed in the present disclosure.Specifically: SGF (Simulated gastric fluids, pH=1.8), FeSSIF (Fed statesimulated intestinal fluids, pH=5.0), FaSSIF (Fasted state simulatedintestinal fluids, pH=6.5), and pure water were used.

Form CS1 and Form CS9 of the present disclosure were suspended into SGF,FeSSIF, FaSSIF and H₂O to obtain saturated solutions. The solutions weresampled at fixed time points. Concentrations of the saturated solutionswere measured by HPLC to measure the kinetic solubility of Form CS1 andForm CS9. The results are listed in Table 11 and 12.

TABLE 11 Kinetic solubility of Form CS1 Solubility(mg/mL) Solvent media1 h 4 h 24 h SGF 0.0031 0.0031 0.0030 FeSSIF 0.19 0.19 0.16 FaSSIF 0.0630.050 0.027 H₂O 0.014 0.026 0.016

TABLE 12 Kinetic solubility of Form CS9 Solubility(mg/mL) Solvent media1 h 4 h 24 h SGF 0.0039 0.0042 0.0093 FeSSIF 0.15 0.17 0.14 FaSSIF 0.0580.081 0.081 H2O 0.020 0.038 0.060

Solubility is one of the key properties of drug substance, whichdirectly affects the absorption of drugs in human body. The solubilityof different crystalline forms may be remarkably different, and the invivo absorption dynamics may also change, which results in differentbioavailability and ultimately affects the clinical safety and efficacyof drugs.

Compound (I) is a poorly water-soluble drug. For poorly water-solubledrug, increasing solubility is even more important. Increase insolubility is conducive to increasing the bioavailability of drugs,thereby increasing the possibility of a successful drug products. Inaddition, the drug dose reduction without affecting efficacy is possibledue to higher solubility, thereby reducing the side effects andimproving the safety of drugs.

Form CS1 and Form CS9 of the present disclosure have good solubility inSGF, FeSSIF, FaSSIF and pure water, providing guarantee for the gooddissolution of the crystalline drug substance in the drug products,which is beneficial to the in vivo absorption of active ingredients indrugs, achieving ideal bioavailability and efficacy.

Example 12: Flowability of Form CS1 and Form CS9

According to the United States Pharmacopeia (USP) 1174, flowability ofForm CS1 and Form CS9 of the present disclosure were evaluated by thecompressibility index. The bulk density, tapped density of Form CS1 andForm CS9 were tested, and compressibility index was calculated. Theresults are listed in Table 13.

-   -   1. Test method:    -   Samples: Form CS1 and Form CS9    -   Instrument: Tap density tester    -   Utensil: 5 mL measuring cylinder    -   Number of tapping: 500 times    -   Bulk density=m/v_(Q) (Mass/volume before tapping)    -   Tapped density=m/v_(t)(Mass/volume after tapping)    -   2. Calculation formula: compressibility index (%)=(tapped        density−bulk density)/tapped density×100%    -   3. Test results:

TABLE 13 Bulk density Tapped density Compressibility Form (g/mL) (g/mL)index (%) Flowability CS1 0.15 0.19 21 Passable CS9 0.11 0.14 21Passable * Scale of flowability (according to US Pharmacopoeia 1174):compressibilityindex ≤10%, excellent flowability; 11%~15%, goodflowability; 16%-20%, fair flowability; 21%-25%, passable flowability;26%~31%, poor flowability; 32%~37%, very poor flowability; >38%,extremely poor flowability.

The results show that the flowability of Form CS1 and Form CS9 of thepresent disclosure meets the requirements for formulation development,ensures the blend uniformity and content uniformity of the drugproducts, reduces the weight variation of the drug products and improvesproduct quality, which is suitable for medicinal use.

Example 13: Study of Form CS1 and Form CS9 in Drug Products

1. Preparation of GSK1278863 tablets:

Form CS1 or Form CS9 of GSK1278863, microcrystalline cellulose,croscarmellose sodium and magnesium stearate were weighed according toformulation in Table 14 and blended for 2 minutes. The tablets wereprepared using a manual tablet press at 5 KN±1 KN pressure with φ7 mmround tooling. The tablets tablet weight is 100 mg±1 mg. The obtainedtablets were packed in 35 cc HDPE bottles (one tablet per bottle) with 1g desiccant. The bottles were sealed with a sealer. The crystalline formof Form CS1 and Form CS9 drug substance doesn't change before and afterformulation process.

TABLE 14 Component Quantity (mg/unit) Mass ratio (%) Form CS1 or FormCS9 12.50 12.50 Microcrystalline Cellulose 81.50 81.50 CroscarmelloseSodium 5.00 5.00 Magnesium Stearate 1.00 1.00 Total 100 100

2. In vitro dissolution profile:

In vitro dissolution test was performed on obtained tablets anddissolution method according to Chinese Pharmacopoeia 2015<0931> wasused. The conditions are as follows:

-   -   Medium: pH=6.8 phosphate buffer solution+1% sodium dodecyl        sulfate aqueous solution    -   Method: Paddle    -   Volume: 900 mL    -   Speed: 75 rpm    -   Temperature: 37° C.

Dissolution results of Form CS1 are presented in Table 15 and FIG. 19 .The results show that the average dissolution of Form CS1 drug productsreaches 67.7% at 10 minutes, and the average dissolution reaches 95.2%at 60 minutes, which indicate that Form CS1 drug products possessesfavorable dissolution, and fast dissolution rate.

Dissolution is a prerequisite for absorption. Good in vitro dissolutionallows drugs to have a higher area under the curve (AUC) in the body,that is, higher in vivo absorption and better in vivo exposure, therebyimproving drug's bioavailability and efficacy. High dissolution ratemakes the drug have a shorter peak time (T_(max)) and a higher peakconcentration (C_(max)) in the body, which is beneficial for the drug toachieve peak plasma concentration quickly after administration, thusensuring rapid drug action.

TABLE 15 Cumulative drug release (%) Time(min) Form CS1  5 0.0  10 67.7 20 75.8  30 84.8  45 90.1  60 95.2  90 98.3 120 102.5

3. Stability of Form CS1 and Form CS9 in drug products:

The above obtained tablets were stored under 40° C./675% RH conditionfor 1 month. After testing, the crystalline form of Form CS1 and FormCS9 drug substance in the drug products does not change. The results areshown in Table 16, which indicate that Form CS1 and Form CS9 have goodstability in the drug products.

TABLE 16 Stability of Form CS1 and Form CS9 in drug products Crystallineforms of API Sample Condition Time after storage Tablets with 40° C./75%RH 1 month Form CS1 Form CS1 Tablets with 40° C./75% RH 1 month Form CS9Form CS9

Form CS1 and Form CS9 of the present disclosure have good stability inthe drug products. Form CS1 and Form CS9 don't readily convert to othercrystal forms during the formulation and storage process, ensuring theconsistent and controllable quality of drug products.

The examples described above are only for illustrating the technicalconcepts and features of the present disclosure, and intended to makethose skilled in the art being able to understand the present disclosureand thereby implement it, and should not be concluded to limit theprotective scope of this disclosure. Any equivalent variations ormodifications according to the spirit of the present disclosure shouldbe covered by the protective scope of the present disclosure.

What is claimed is:
 1. A composition of crystalline form CS1 ofN-[(1,2-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine,wherein the X-ray powder diffraction pattern of form CS1 showscharacteristic peaks at 2theta values of 6.4° 00.2°, 7.5°±0.2°, and7.9°±0.2° using CuKα radiation, and wherein the content of othercrystalline forms is less than 10% (w/w).
 2. The composition accordingto claim 1, wherein the X-ray powder diffraction pattern of Form CS1shows one or more characteristic peaks at 2theta values of 17.2°±0.2°,21.0°±0.2°, 24.0°±0.2°, and 19.3°±0.2° using CuKα radiation.
 3. Thecomposition according to claim 1, wherein the X-ray powder diffractionpattern of Form CS1 is substantially as depicted in FIG. 1 .
 4. Thecomposition according to claim 1, wherein the Form CS1 has anendothermic peak at around 242° C. as measured by differential scanningcalorimetry using a heating rate of 10° C. min and a purge gas ofnitrogen.
 5. The composition according to claim 1, wherein the chemicalpurity of Form CS1 is higher than 99%.
 6. The composition according toclaim 5, wherein the chemical impurity remains substantially unchangedwhen the composition is stored under conditions of 60° C. 75% RH for 3months.
 7. A composition of crystalline form CS9 ofN-[(1,2-dicyclohexylhexahydro-2,4,6-trioxo-5-pyrimidinyl)carbonyl]-glycine,wherein the X-ray powder diffraction pattern of form CS9 showscharacteristic peaks at 2theta values of 4.6°±0.2°, 6.6°±0.2°, and21.1°±0.2° using CuKα radiation, and wherein the content of othercrystalline forms is less than 10% (w/w).
 8. The composition accordingto claim 7, wherein the X-ray powder diffraction pattern shows one ormore characteristic peaks at 2theta values of 9.4°±0.2°, 20.2°±0.2°, and24.2°±0.2° using CuKα radiation.
 9. The composition according to claim7, wherein the X-ray powder diffraction pattern of Form CS9 issubstantially as depicted in FIG. 6 .
 10. The composition according toclaim 7, wherein the Form 9 has a first endothermic peak at around 145°C. and a second endothermic peak at around 237° C. as measured bydifferential scanning calorimetry using a heating rate of 10° C. min anda purge gas of nitrogen.
 11. The composition according to claim 7,wherein the chemical purity of Form CS9 is higher than 99%.
 12. Thecomposition according to claim 11, wherein the chemical impurity remainssubstantially unchanged when the composition is stored under conditionsof 60° C./5% RH for 1 month.
 13. A pharmaceutical composition, whereinsaid pharmaceutical composition comprises a therapeutically effectiveamount of the composition of crystalline form CS1 according to claim 1,and pharmaceutically acceptable carriers, diluents or excipients.
 14. Apharmaceutical composition according to claim 13, wherein thecrystalline form does not change following one-month storage at 40°C./5% RH.
 15. A method of treating anemia, comprising administering to asubject in need thereof a therapeutically effective amount of thecomposition of crystalline form CS1 according to claim
 1. 16. Apharmaceutical composition, wherein said pharmaceutical compositioncomprises a therapeutically effective amount of the composition ofcrystalline form CS9 according to claim 7, and pharmaceuticallyacceptable carriers, diluents or excipients.
 17. A pharmaceuticalcomposition according to claim 16, wherein the crystalline form does notchange following one-month storage at 40° C./5% RH.
 18. A method oftreating anemia, comprising administering to a subject in need thereof atherapeutically effective amount of the crystalline form CS9 accordingto claim 7.